Mentions:
To assess effects of copper chelator on CoCl2-induced EMT of MCF-7 cells, TEPA, a specific copper chelator, was used to deplete the copper in culture medium. After 24-h treatment with TEPA (50 μM), few elongated and spindle-like mesenchyme-like cells were observed in either control or TEPA-treated groups (Fig. 3A). In contrast, cells under hypoxia-induced by CoCl2 (200 μM) exhibited epithelial cobblesone-like morphology (Fig. 3A). Similar results were obtained by quantitative evaluation of cell polarity. Copper depletion with TEPA showed 10% cells with polarity value above 2, which was similar to 6% cells with polarity value above 2 in control group (Fig. 3B). Cytoskeleton rearrangement and the formation of stress fibers were observed under oil objective (Fig. 3C). After 24 h treatment with CoCl2, cells exhibited typical fibroblast-like phenotype with actin filaments bundled into thick contractile stress fibers at the ventral cell surface, whereas cells treated with both CoCl2 and TEPA showed cortical thin bundles of actin filaments that were typical in control group. Furthermore, compared to cells treated with CoCl2, TEPA treatment reduced both nuclear (Fig. 4A,B) and total (Fig. 4D,E) accumulation of HIF1-α; significantly increased the expression of E-cadherin, an epithelial marker (Fig. 4D,E); and significantly decreased the expression of vimentin and fibronectin, markers of mesenchymal cells, at both mRNA and protein levels (Fig. 4C–E) as evaluated by both real-time PCR and Western blotting, respectively. Moreover, immunofluorescence data (Fig. 5) and quantitative analysis (Fig. S1) also demonstrated TEPA treatment increased the expression of E-cadherin and down-regulated expression of vimentin. Taken together, these results suggest that TEPA inhibited CoCl2-induced EMT of MCF-7 cells.

Mentions:
To assess effects of copper chelator on CoCl2-induced EMT of MCF-7 cells, TEPA, a specific copper chelator, was used to deplete the copper in culture medium. After 24-h treatment with TEPA (50 μM), few elongated and spindle-like mesenchyme-like cells were observed in either control or TEPA-treated groups (Fig. 3A). In contrast, cells under hypoxia-induced by CoCl2 (200 μM) exhibited epithelial cobblesone-like morphology (Fig. 3A). Similar results were obtained by quantitative evaluation of cell polarity. Copper depletion with TEPA showed 10% cells with polarity value above 2, which was similar to 6% cells with polarity value above 2 in control group (Fig. 3B). Cytoskeleton rearrangement and the formation of stress fibers were observed under oil objective (Fig. 3C). After 24 h treatment with CoCl2, cells exhibited typical fibroblast-like phenotype with actin filaments bundled into thick contractile stress fibers at the ventral cell surface, whereas cells treated with both CoCl2 and TEPA showed cortical thin bundles of actin filaments that were typical in control group. Furthermore, compared to cells treated with CoCl2, TEPA treatment reduced both nuclear (Fig. 4A,B) and total (Fig. 4D,E) accumulation of HIF1-α; significantly increased the expression of E-cadherin, an epithelial marker (Fig. 4D,E); and significantly decreased the expression of vimentin and fibronectin, markers of mesenchymal cells, at both mRNA and protein levels (Fig. 4C–E) as evaluated by both real-time PCR and Western blotting, respectively. Moreover, immunofluorescence data (Fig. 5) and quantitative analysis (Fig. S1) also demonstrated TEPA treatment increased the expression of E-cadherin and down-regulated expression of vimentin. Taken together, these results suggest that TEPA inhibited CoCl2-induced EMT of MCF-7 cells.

Bottom Line:
Using CoCl2-induced EMT of human breast carcinoma MCF-7 cells, we found that TEPA, a copper chelator, inhibited EMT-like cell morphology and cytoskeleton arrangement triggered by CoCl2; decreased the expression of vimentin and fibronectin, markers typical of EMT; inhibited HIF-1 activation and HIF1-α accumulation in nuclear; and down-regulated the expression of hypoxia-associated transcription factors, Snail and Twist1.Immunohistochemical analysis of the xenograft further demonstrated that TEPA administration significantly inhibited tumor angiogenesis, down-regulated hypoxia-induced transcription factors, Snail and Twist1, leading to decreased transactivation of EMT-associated marker genes, vimentin and fibronectin.These results indicate that TEPA inhibits CoCl2-induced EMT most likely via HIF1-α-Snail/Twist signaling pathway, and copper depletion may be exploited as a therapeutic for breast cancer.